Antisense oligonucleotide inhibition of ras

ABSTRACT

Compositions and methods are provided for the modulation of ras expression. Oligonucleotides are provided which are targeted to nucleic acids encoding human ras. Oligonucleotides specifically hybridizable with mRNA encoding human H-ras, Ki-ras and N-ras are provided. Such oligonucleotides can be used for therapeutics and diagnostics as well as for research purposes. Methods are also disclosed for modulating ras gene expression in cells and tissues using the oligonucleotides provided, and for specific modulation of expression of activated ras. Methods for diagnosis, detection and treatment of conditions associated with ras are also disclosed.

This application is a continuation-in-part of U.S. patent applicationNo. 08/411,734, filed Apr. 3, 1995 which is a U.S. national phaseapplication of PCT/US93/09346, filed Oct. 1, 1993, which is acontinuation-in-part and foreign filing of U.S. patent application No.958,134, filed Oct. 5, 1992 now abandoned, and U.S. patent applicationNo. 08/007,996, filed Jan. 21, 1993 now abandoned, all of which areassigned to the assignee of the present invention and are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to compositions and methods for the inhibition ofexpression of ras, a naturally occurring protein which occasionallyconverts to an activated form that has been implicated in tumorformation. Antisense oligonucleotides targeted to H-, Ki- and N-ras areprovided. This invention is further directed to the detection of bothnormal and activated forms of the ras gene in cells and tissues, and canform the basis for research reagents and kits both for research anddiagnosis. Furthermore, this invention is directed to prevention andtreatment of conditions associated with ras.

BACKGROUND OF THE INVENTION

Alterations in the cellular genes which directly or indirectly controlcell growth and differentiation are considered to be the main cause ofcancer. There are some thirty families of genes, called oncogenes, whichare implicated in human tumor formation. Members of one such family, theras gene family, are frequently found to be mutated in human tumors. Intheir normal state, proteins produced by the ras genes are thought to beinvolved in normal cell growth and maturation. Mutation of the ras gene,causing an amino acid alteration at one of three critical positions inthe protein product, results in conversion to a form which is implicatedin tumor formation. A gene having such a mutation is said to be "mutant"or "activated." Unmutated ras is called "wild-type" or "normal" ras. Itis thought that such a point mutation leading to ras activation can beinduced by carcinogens or other environmental factors. Over 90% ofpancreatic adenocarcinomas, about 50% of adenomas and adenocarcinomas ofthe colon, about 50% of adenocarcinomas of the lung and carcinomas ofthe thyroid, and a large fraction of malignancies of the blood such asacute myeloid leukemia and myelodysplastic syndrome have been found tocontain activated ras oncogenes. Overall, some 10 to 20% of human tumorshave a mutation in one of the three ras genes (H-ras, Ki-ras, or N-ras).

It is presently believed that inhibiting expression of activatedoncogenes in a particular tumor cell might force the cell back into morenormal growth. For example, Feramisco et al., Nature 1985, 314, 639-642,demonstrated that if cells transformed to a malignant state with anactivated ras gene are microinjected with antibody which binds to theprotein product of the ras gene, the cells slow their rate ofproliferation and adopt a more normal appearance. This has beeninterpreted as support for the involvement of the product of theactivated ras gene in the uncontrolled growth typical of cancer cells.

There is a great desire to provide compositions of matter which canmodulate the expression of ras, and particularly to provide compositionsof matter which specifically modulate the expression of activated ras.It is greatly desired to provide methods of diagnosis and detection ofnucleic acids encoding ras in animals. It is also desired to providemethods of diagnosis and treatment of conditions arising from rasactivation. In addition, improved research kits and reagents fordetection and study of nucleic acids encoding ras are desired.

Inhibition of oncogene expression has been accomplished using retroviralvectors or plasmid vectors which express a 2-kilobase segment of theKi-ras protooncogene RNA in antisense orientation. Mukhopadhyay, T. etal. (1991) Cancer Research 51, 1744-1748; PCT Patent ApplicationPCT/US92/01852 (WO 92/15680); Georges, R. N. et al. (1993) CancerResearch, 53, 1743-1746.

Antisense oligonucleotide inhibition of oncogenes has proven to be auseful tool in understanding the roles of various oncogene families.Antisense oligonucleotides are small oligonucleotides which arecomplementary to the "sense" or coding strand of a given gene, and as aresult are also complementary to, and thus able to stably andspecifically hybridize with, the mRNA transcript of the gene. Holt etal., Mol. Cell Biol. 1988, 8, 963-973, have shown that antisenseoligonucleotides hybridizing specifically with mRNA transcripts of theoncogene c-myc, when added to cultured HL60 leukemic cells, inhibitproliferation and induce differentiation. Anfossi et al., Proc. Natl.Acad. Sci. 1989, 86, 3379-3383, have shown that antisenseoligonucleotides specifically hybridizing with mRNA transcripts of thec-myb oncogene inhibit proliferation of human myeloid leukemia celllines. Wickstrom et al., Proc. Nat. Acad. Sci. 1988, 85, 1028-1032, haveshown that expression of the protein product of the c-myc oncogene aswell as proliferation of HL60 cultured leukemic cells are inhibited byantisense oligonucleotides hybridizing specifically with c-myc mRNA.U.S. Pat. No: 4,871,838 (Bos et al.) discloses oligonucleotidescomplementary to a mutation in codon 13 of N-ras to detect saidmutation. U.S. Pat. No: 4,871,838 (Bos et al.) discloses moleculesuseful as probes for detecting a mutation in DNA which encodes a rasprotein.

In all these cases, instability of unmodified oligonucleotides has beena major problem, as they are subject to degradation by cellular enzymes.PCT/US88/01024 (Zon et al.) discloses phosphorothioate oligonucleotideshybridizable to the translation initiation region of the amplified c-myconcogene to inhibit HL-60 leukemia cell growth and DNA synthesis inthese cells. Tidd et al., Anti-Cancer Drug Design 1988, 3, 117-127,evaluated methylphosphonate antisense oligonucleotides hybridizingspecifically to the activated N-ras oncogene and found that while theywere resistant to biochemical degradation and were nontoxic in culturedhuman HT29 cells, they did not inhibit N-ras gene expression and had noeffect on these cells. Chang et al. showed that both methylphosphonateand phosphorothioate oligonucleotides hybridizing specifically to mRNAtranscripts of the mouse Balb-ras gene could inhibit translation of theprotein product of this gene in vitro. Chang et al., Anti-Cancer DrugDesign 1989, 4, 221-232; Brown et al., Oncogene Research 1989, 4,243-252. It was noted that TM was not well correlated with antisenseactivity of these oligonucleotides against in vitro translation of theras p21 protein product. Because the antisense oligonucleotides used byChang et al. hybridize specifically with the translation initiationregion of the ras gene, they are not expected to show any selectivityfor activated ras and the binding ability of these oligonucleotides tonormal (wild-type) vs. mutated (activated) ras genes was not compared.

Helene and co-workers have demonstrated selective inhibition ofactivated (codon 12 G→T transition) H-ras mRNA expression using a 9-merphosphodiester linked to an acridine intercalating agent and/or ahydrophobic tail. This compound displayed selective targeting of mutantras message in both Rnase H and cell proliferation assays at lowmicromolar concentrations. Saison-Behmoaras, T. et al., EMBO J. 1991,10, 1111-1118. Chang and co-workers disclose selective targeting ofmutant H-ras message; this time the target was H-ras codon 61 containingan A→T transversion and the oligonucleotide employed was either an11-mer methylphosphonate or its psoralen derivative. These compounds,which required concentrations of 7.5-150 μM for activity, were shown byimmunoprecipitation to selectively inhibit mutant H-ras p21 expressionrelative to normal p21. Chang et al., Biochemistry 1991, 30, 8283-8286.

SUMMARY OF THE INVENTION

The present invention relates to antisense oligonucleotides which aretargeted to human ras, and methods of using them. More specifically, thepresent invention provides oligonucleotides which are targeted to mRNAencoding human H-ras, Ki-ras and N-ras and which are capable ofinhibiting ras expression. Oligonucleotides targeted to a 5'untranslated region, translation initiation site, coding region or 3'untranslated region of human N-ras are provided. Methods of modulatingras expression, of inhibiting the proliferation of cancer cells and oftreating conditions associated with ras are provided. These methodsemploy the oligonucleotides of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H are a series of 8 panels showing inhibition of ras in adose-dependent manner. Solid lines are activity against wild-type(normal) ras, dotted lines show activity against activated (mutant) ras.

FIG. 2 is a bar graph showing antisense activities of a uniform deoxyphosphorothioate and shortened chimeric oligonucleotides againstras-luciferase.

FIG. 3 is a line graph showing correlation between antisense activityand ability to activate RNAse H as a function of deoxy gap length usingphosphorothioate 2'-O-methyl oligonucleotides targeted against ras.

FIG. 4 is a line graph showing anti-tumor activity of ISIS 2503 againstA549 human cell tumors in nude mice.

FIG. 5 is a line graph showing anti-tumor activity of ras oligo ISIS2503, administered with cationic lipid, against A549 human cell tumorsin nude mice.

FIG. 6 is a bar graph showing antisense inhibition of Ki-ras mRNAexpression in three human colon carcinoma cell lines, Calul, SW480 andSW620.

FIG. 7 is a bar graph showing inhibition of SW480 human carcinoma cellline proliferation by Ki-ras specific oligonucleotides ISIS 6957 andISIS 6958.

FIG. 8 is a bar graph showing reduction of H-ras mRNA levels by 2'-MOEanalogs of ISIS 2503 (SEQ ID NO: 2). Black bars: 150 nM oligonucleotidedose; Diagonal hatched bars: 50 nM dose; horizontal hatched bars: 15 nMdose.

FIG. 9 is a bar graph showing reduction of H-ras mRNA levels by MMIanalogs of ISIS 2503 (SEQ ID NO: 2). Black bars: 500 nM oligonucleotidedose; Diagonal hatched bars: 100 nM dose; horizontal hatched bars: 50 nMdose.

FIG. 10 is a bar graph showing reduction of N-ras mRNA levels byoligonucleotides 14686-14694, 14677 and 14678. Black bars: 400 nMoligonucleotide dose; Diagonal hatched bars: 200 nM dose; horizontalhatched bars: 100 nM dose.

DETAILED DESCRIPTION OF THE INVENTION

Malignant tumors develop through a series of stepwise, progressivechanges that lead to the loss of growth control characteristic of cancercells, i.e., continuous unregulated proliferation, the ability to invadesurrounding tissues, and the ability to metastasize to different organsites. Carefully controlled in vitro studies have helped define thefactors that characterize the growth of normal and neoplastic cells andhave led to the identification of specific proteins that control cellgrowth and differentiation. In addition, the ability to study celltransformation in carefully controlled, quantitative in vitro assays hasled to the identification of specific genes capable of inducing thetransformed cell phenotype. Such cancer-causing genes, or oncogenes, arebelieved to acquire transformation-inducing properties through mutationsleading to changes in the regulation of expression of their proteinproducts. In some cases such changes occur in non-coding DNA regulatorydomains, such as promoters and enhancers, leading to alterations in thetranscriptional activity of oncogenes, resulting in over- orunder-expression of their gene products. In other cases, gene mutationsoccur within the coding regions of oncogenes, leading to the productionof altered gene products that are inactive, overactive, or exhibit anactivity that is different from the normal (wild-type) gene product.

To date, more than 30 cellular oncogene families have been identified.These genes can be categorized on the basis of both their subcellularlocation and the putative mechanism of action of their protein products.The ras oncogenes are members of a gene family which encode relatedproteins that are localized to the inner face of the plasma membrane.ras proteins have been shown to be highly conserved at the amino acidlevel, to bind GTP with high affinity and specificity, and to possessGTPase activity. Although the cellular function of ras gene products isunknown, their biochemical properties, along with their significantsequence homology with a class of signal-transducing proteins known asGTP binding proteins, or G proteins, suggest that ras gene products playa fundamental role in basic cellular regulatory functions relating tothe transduction of extracellular signals across plasma membranes.

Three ras genes, designated H-ras, Ki-ras, and N-ras, have beenidentified in the mammalian genome. Mammalian ras genes acquiretransformation-inducing properties by single point mutations withintheir coding sequences. Mutations in naturally occurring ras oncogeneshave been localized to codons 12, 13, and 61. The sequences of H-ras,Ki-ras and N-ras are known. Capon et al., Nature 302 1983, 33-37; Kahnet al., Anticancer Res. 1987, 7, 639-652; Hall and Brown, Nucleic AcidsRes. 1985, 13, 5255-5268. The most commonly detected activating rasmutation found in human tumors is in codon 12 of the H-ras gene in whicha base change from GGC to GTC results in a glycine-to-valinesubstitution in the GTPase regulatory domain of the ras protein product.Tabin, C. J. et al., Nature 1982, 300, 143-149; Reddy, P. E. et al.,Nature 1982, 300, 149-152; Taparowsky, E. et al., Nature 1982, 300,762-765. This single amino acid change is thought to abolish normalcontrol of ras protein function, thereby converting a normally regulatedcell protein to one that is continuously active. It is believed thatsuch deregulation of normal ras protein function is responsible for thetransformation from normal to malignant growth. It is therefore believedthat inhibition of ras expression is useful in treatment and/orprevention of malignant conditions, i.e., cancer and otherhyperproliferative conditions.

The H-ras gene has recently been implicated in a serious cardiacarrhythmia called long Q-T syndrome, a hereditary condition which oftencauses sudden death if treatment is not given immediately. Frequently,there are no symptoms prior to the onset of the erratic heartbeat.Whether the H-ras gene is precisely responsible for long Q-T syndrome isunclear. However, there is an extremely high correlation betweeninheritance of this syndrome and the presence of a particular variant ofthe chromosome 11 region surrounding the H-ras gene. Therefore, theH-ras gene is a useful indicator of increased risk of sudden cardiacdeath due to the long Q-T syndrome.

N-ras was first identified as an oncogene in gene transfer experiments.Hall et al. Nature 1983, 303: 396-400. Its activation was characterizedby Taparowsky et al. Cell 1983 34: 581-6. Activated N-ras is found inmany hematologic neoplasms and solid tumors, suggesting a role for N-rasin the development or maintenance of hyperproliferative conditions.

The present invention provides oligonucleotides for inhibition of humanras gene expression. Such oligonucleotides specifically hybridize withselected DNA or mRNA deriving from a human ras gene. The invention alsoprovides oligonucleotides for selective inhibition of expression of themutant form of ras. This relationship between an oligonucleotide and itscomplementary nucleic acid target to which it hybridizes is commonlyreferred to as "antisense". "Targeting" an oligonucleotide to a chosennucleic acid target, in the context of this invention, is a multistepprocess. The process usually begins with identifying a nucleic acidsequence whose function is to be modulated. This may be, for example, acellular gene (or mRNA made from the gene) whose expression isassociated with a particular disease state, or a foreign nucleic acidfrom an infectious agent. In the present invention, the target is anucleic acid encoding ras; in other words, the ras gene or mRNAexpressed from the ras gene. The targeting process also includesdetermination of a site or sites within the nucleic acid sequence forthe oligonucleotide interaction to occur such that the desiredeffect--modulation of gene expression--will result. Once the target siteor sites have been identified, oligonucleotides are chosen which aresufficiently complementary to the target, i.e., hybridize sufficientlywell and with sufficient specificity, to give the desired modulation.

In the context of this invention "modulation" means either inhibition orstimulation. Inhibition of ras gene expression is presently thepreferred form of modulation. This modulation can be measured in wayswhich are routine in the art, for example by Northern blot assay of mRNAexpression or Western blot assay of protein expression as taught in theexamples of the instant application. Effects on cell proliferation ortumor cell growth can also be measured, as taught in the examples of theinstant application. "Hybridization", in the context of this invention,means hydrogen bonding, also known as Watson-Crick base pairing, betweencomplementary bases, usually on opposite nucleic acid strands or tworegions of a nucleic acid strand. Guanine and cytosine are examples ofcomplementary bases which are known to form three hydrogen bonds betweenthem. Adenine and thymine are examples of complementary bases which formtwo hydrogen bonds between them. "Specifically hybridizable" and"complementary" are terms which are used to indicate a sufficient degreeof complementarity such that stable and specific binding occurs betweenthe DNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget interferes with the normal function of the target molecule tocause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment or, in the case of in vitro assays,under conditions in which the assays are conducted.

In preferred embodiments of this invention, oligonucleotides areprovided which are targeted to mRNA encoding H-ras, Ki-ras or N-ras. Inaccordance with this invention, persons of ordinary skill in the artwill understand that mRNA includes not only the coding region whichcarries the information to encode a protein using the three lettergenetic code, including the translation start and stop codons, but alsoassociated ribonucleotides which form a region known to such persons asthe 5'-untranslated region, the 3'-untranslated region, the 5' capregion, intron regions and intron/exon or splice junctionribonucleotides. Thus, oligonucleotides may be formulated in accordancewith this invention which are targeted wholly or in part to theseassociated ribonucleotides as well as to the coding ribonucleotides. Inpreferred embodiments, the oligonucleotide is targeted to a translationinitiation site (AUG codon) or sequences in the coding region, 5'untranslated region or 3'-untranslated region of the ras mRNA. Thefunctions of messenger RNA to be interfered with include all vitalfunctions such as translocation of the RNA to the site for proteintranslation, actual translation of protein from the RNA, splicing ormaturation of the RNA and possibly even independent catalytic activitywhich may be engaged in by the RNA. The overall effect of suchinterference with the RNA function is to cause interference with rasprotein expression.

The present invention provides oligonucleotides for modulation of rasgene expression. Such oligonucleotides are targeted to nucleic acidsencoding ras. As hereinbefore defined, "modulation" means eitherinhibition or stimulation. Inhibition of ras gene expression ispresently the preferred form of modulation.

In the context of this invention, the term "oligonucleotide" refers toan oligomer or polymer of nucleotide or nucleoside monomers consistingof naturally occurring bases, sugars and intersugar (backbone) linkages.The term "oligonucleotide" also includes oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake and increased stability in the presence ofnucleases.

Certain preferred oligonucleotides of this invention are chimericoligonucleotides. "Chimeric oligonucleotides" or "chimeras", in thecontext of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionof modified nucleotides that confers one or more beneficial properties(such as, for example, increased nuclease resistance, increased uptakeinto cells, increased binding affinity for the RNA target) and a regionthat is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of antisense inhibition of gene expression.Consequently, comparable results can often be obtained with shorteroligonucleotides when chimeric oligos are used, compared tophosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art. In one preferred embodiment, a chimericoligonucleotide comprises at least one region modified to increasetarget binding affinity, and, usually, a region that acts as a substratefor RNAse H. Affinity of an oligonucleotide for its target (in thiscase, a nucleic acid encoding ras) is routinely determined by measuringthe Tm of an oligonucleotide/target pair, which is the temperature atwhich the oligonucleotide and target dissociate; dissociation isdetected spectrophotometrically. The higher the Tm, the greater theaffinity of the oligonucleotide for the target. In a more preferredembodiment, the region of the oligonucleotide which is modified toincrease ras mRNA binding affinity comprises at least one nucleotidemodified at the 2' position of the sugar, most preferably a 2'-O-alkyl,2'-O-alkyl-O-alkyl or 2'-fluoro-modified nucleotide. Such modificationsare routinely incorporated into oligonucleotides and theseoligonucleotides have been shown to have a higher Tm (i.e., highertarget binding affinity) than 2'-deoxyoligonucleotides against a giventarget. The effect of such increased affinity is to greatly enhanceantisense oligonucleotide inhibition of ras gene expression. RNAse H isa cellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes;activation of this enzyme therefore results in cleavage of the RNAtarget, and thus can greatly enhance the efficiency of antisenseinhibition. Cleavage of the RNA target can be routinely demonstrated bygel electrophoresis. In another preferred embodiment, the chimericoligonucleotide is also modified to enhance nuclease resistance. Cellscontain a variety of exo- and endo-nucleases which can degrade nucleicacids. A number of nucleotide and nucleoside modifications have beenshown to make the oligonucleotide into which they are incorporated moreresistant to nuclease digestion than the native oligodeoxynucleotide.Nuclease resistance is routinely measured by incubating oligonucleotideswith cellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotide remaining over time, usually by gelelectrophoresis. Oligonucleotides which have been modified to enhancetheir nuclease resistance survive intact for a longer time thanunmodified oligonucleotides. A variety of oligonucleotide modificationshave been demonstrated to enhance or confer nuclease resistance.Oligonucleotides which contain at least one phosphorothioatemodification are presently more preferred. In some cases,oligonucleotide modifications which enhance target binding affinity arealso, independently, able to enhance nuclease resistance. A discussionof antisense oligonucleotides and some desirable modifications can befound in De Mesmaeker et al. Acc. Chem. Res. 1995, 28:366-374.

Specific examples of some preferred oligonucleotides envisioned for thisinvention include those containing modified backbones, for example,phosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are oligonucleotideswith phosphorothioate backbones and those with heteroatom backbones,particularly CH₂ --NH--O--CH₂, CH₂ --N(CH₃)--O--CH₂ known as amethylene(methylimino) or MMI backbone!, CH₂ --O--N(CH₃)--CH₂, CH₂--N(CH₃)--N(CH₃)--CH₂ and O--N(CH₃)--CH₂ --CH₂ backbones, wherein thenative phosphodiester backbone is represented as O--P--O--CH₂). Theamide backbones disclosed by De Mesmaeker et al. Acc. Chem. Res. 1995,28:366-374) are also preferred. Also preferred are oligonucleotideshaving morpholino backbone structures (Summerton and Weller, U.S. Pat.No. 5,034,506). In other preferred embodiments, such as the peptidenucleic acid (PNA) backbone, the phosphodiester backbone of theoligonucleotide is replaced with a polyamide backbone, the nucleobasesbeing bound directly or indirectly to the aza nitrogen atoms of thepolyamide backbone (Nielsen et al. Science 1991, 254, 1497).

Oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2' position: OH, SH, SCH₃, F, OCN, OCH₃ OCH₃, OCH₃ O(CH₂)_(n) CH₃,O(CH₂)_(n) NH₂ or O(CH₂)_(n) CH₃ where n is from 1 to about 10; C₁ toC₁₀ lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl oraralkyl; Cl; Br; CN; CF₃ ; OCF₃ ; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; SOCH₃ ; SO₂ CH₃ ; ONO₂ ; NO₂ ; N₃ ; NH₂ ; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. A preferredmodification includes 2'-methoxyethoxy 2'--O--CH₂ CH₂ OCH₃, also knownas 2'-O-(2-methoxyethyl)! (Martin et al., Helv. Chim. Acta, 1995, 78,486). Other preferred modifications include 2'-methoxy (2'--O--CH₃),2'-propoxy (2'--OCH₂ CH₂ CH₃) and 2'-fluoro (2'-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3' position of the sugar on the 3'terminal nucleotide and the 5' position of 5' terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutyls inplace of the pentofuranosyl group.

Oligonucleotides may also include, additionally or alternatively,nucleobase (often referred to in the art simply as "base") modificationsor substitutions. As used herein, "unmodified" or "natural" nucleobasesinclude adenine (A), guanine (G), thymine (T), cytosine (C) and uracil(U). Modified nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2'deoxycytosine and often referred to in the artas 5-me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosylHMC, as well as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N⁶ (6-aminohexyl)adenine and2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co.,San Francisco, 1980, pp75-77; Gebeyehu, G., et al. Nucl. Acids Res.1987, 15:4513). A "universal" base known in the art, e.g., inosine, maybe included. 5-me-C substitutions have been shown to increase nucleicacid duplex stability by 0.6°-1.2° C. (Sanghvi, Y. S., in Crooke, S. T.and Lebleu, B., eds., Antisense Research and Applications, CRC Press,Boca Raton, 1993, pp. 276-278) and are presently preferred basesubstitutions.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety, a cholesteryl moiety (Letsingeret al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553), cholic acid(Manoharan et al. Bioorg. Med. Chem. Let. 1994, 4, 1053), a thioether,e.g., hexyl-S-tritylthiol (Manoharan et al. Ann. N.Y. Acad. Sci. 1992,660, 306; Manoharan et al. Bioorg. Med. Chem. Let. 1993, 3, 2765), athiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al. EMBO J. 1991, 10, 111; Kabanov et al. FEBS Lett. 1990, 259, 327;Svinarchuk et al. Biochimie 1993, 75, 49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.Tetrahedron Lett. 1995, 36, 3651; Shea et al. Nucl. Acids Res. 1990, 18,3777), a polyamine or a polyethylene glycol chain (Manoharan et al.Nucleosides & Nucleotides 1995, 14, 969), or adamantane acetic acid(Manoharan et al. Tetrahedron Lett. 1995, 36, 3651). Oligonucleotidescomprising lipophilic moieties, and methods for preparing sucholigonucleotides are known in the art, for example, U.S. Pat. No.5,138,045, No. 5,218,105 and No. 5,459,255.

The oligonucleotides of the invention may be provided as prodrugs, whichcomprise one or more moieties which are cleaved off, generally in thebody, to yield an active oligonucleotide. One example of a prodrugapproach is described by Imbach et al. in WO Publication 94/26764.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even atwithin a single nucleoside within an oligonucleotide. The presentinvention also includes oligonucleotides which are chimericoligonucleotides as hereinbefore defined.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the routineer. It is also well known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives. It is also well known touse similar techniques and commercially available modified amidites andcontrolled-pore glass (CPG) products such as biotin, fluorescein,acridine or psoralen-modified amidites and/or CPG (available from GlenResearch, Sterling Va.) to synthesize fluorescently labeled,biotinylated or other modified oligonucleotides such ascholesterol-modified oligonucleotides.

The oligonucleotides in accordance with this invention preferablycomprise from about 8 to about 50 nucleic acid base units. In thecontext of this invention it is understood that this encompassesnon-naturally occurring oligomers as hereinbefore described, having 8 to50 monomers.

The oligonucleotides of this invention can be used in diagnostics,therapeutics and as research reagents and kits. Since theoligonucleotides of this invention hybridize to the ras gene, sandwichand other assays can easily be constructed to exploit this fact.Furthermore, since the oligonucleotides of this invention hybridizepreferentially to the mutant (activated) form of the ras oncogene, suchassays can be devised for screening of cells and tissues for rasconversion from wild-type to activated form. Such assays can be utilizedfor differential diagnosis of morphologically similar tumors, and fordetection of increased risk of cancer stemming from ras gene activation.Provision of means for detecting hybridization of oligonucleotide withthe ras gene can routinely be accomplished. Such provision may includeenzyme conjugation, radiolabelling or any other suitable detectionsystems. Kits for detecting the presence or absence of nucleic acidsencoding ras or activated ras may also be prepared.

The following specific descriptions serve to illustrate the inventionand are not intended to limit the scope of the invention:

Antisense Oligonucleotide Inhibition of ras-Luciferase Gene Expression:

A series of antisense phosphorothioate oligonucleotides targeted toeither the H-ras translation initiation codon or the codon-12 pointmutation of activated H-ras were screened using the ras-luciferasereporter gene system described in Examples 2-5. Of this initial series,six oligonucleotides were identified that gave significant andreproducible inhibition of ras-luciferase activity. The base sequences,sequence reference numbers and SEQ ID numbers of these oligonucleotides(all are phosphorothioates) are shown in Table 1.

                  TABLE 1    ______________________________________    OLIGO    REF NO  SEQUENCE               SEQ ID NO:    ______________________________________    2502    CTT-ATA-TTC-CGT-CAT-CGC-TC                                   1    2503    TCC-GTC-ATC-GCT-CCT-CAG-GG                                   2    2570    CCA-CAC-CGA-CGG-CGC-CC 3    2571    CCC-ACA-CCG-ACG-GCG-CCC-A                                   4    2566    GCC-CAC-ACC-GAC-GGC-GCC-CAC                                   5    2560    TGC-CCA-CAC-CGA-CGG-CGC-CCA-CC                                   6    ______________________________________

A dose-response experiment was performed in which cells expressingeither the normal ras-luciferase reporter gene or the mutantras-luciferase reporter gene were treated with increasing concentrationsof the phosphorothioate oligonucleotide 2503 (SEQ ID NO: 2). Thiscompound is targeted to the translational initiation codon of H-ras RNAtranscripts. Treatment of cells with this oligonucleotide resulted in adose-dependent inhibition of ras-luciferase activity, displaying IC50values of approximately 50 nM for both the normal and the mutant rastargets. The observation that an oligonucleotide targeted to the rastranslation initiation codon is equally effective in reducing bothmutant and normal ras expression is expected since the two targets haveidentical sequence compositions in the region surrounding the AUGtranslation initiation site.

Another dose-response experiment was performed in which cells weretreated with phosphorothioate oligonucleotide 2570 (SEQ ID NO: 3), acompound that is targeted to the codon-12 point mutation of mutant(activated) H-ras RNA. Treatment of cells with increasing concentrationsof this oligonucleotide resulted in a dose-dependent inhibition ofras-luciferase activity in cells expressing either the mutant form orthe normal form of ras-luciferase. However, oligonucleotide 2570displayed approximately threefold selectivity toward the mutant form ofras-luciferase as compared to the normal form. In fact, 2570 displayedan IC50 value for the mutant form of ras-luciferase of approximately 100nM whereas the same compound displayed in IC50 value of approximately250 nM for the unmutated form.

Cells expressing either the normal form or the mutant form ofras-luciferase were treated with a single dose (0.5 μM) ofoligonucleotide targeted to either the translation initiation codon ofH-ras or the codon-12 point mutation. The antisense phosphorothioateoligonucleotides tested are shown in Table 1. Compound 2503 (SEQ ID NO:2), targeted to the ras translational initiation codon, was mosteffective in inhibiting ras-luciferase activity, giving approximately80% inhibition of both normal and mutant targets. ISIS 2502 gave 30-35%inhibition of both targets. Of the three compounds targeted to thecodon-12 point mutation of activated H-ras, only the 17-meroligonucleotide 2570 (SEQ ID NO: 3) displayed selectivity toward themutated form of ras-luciferase as compared to the normal form, givingapproximately 22% inhibition of the normal target and 68% inhibition ofthe mutant target. ISIS 2571 gave approximately 60% inhibition of bothtargets and ISIS 2566 gave 65-70% inhibition of both targets. Table 2summarizes data obtained with all 13 antisense oligonucleotides targetedto H-ras. A scrambled control oligonucleotide gave no inhibition ofeither mutant or normal ras and a control oligonucleotide (ISIS 2907;SEQ ID NO: 19) complementary to the codon-12 region of normal ras gave70% inhibition of the normal target but had no effect on the mutant ras.Shown for each oligonucleotide is its sequence, region to which it iscomplementary, and its activity in suppressing expression of theras-luciferase fusion protein (given as IC50, the concentration in nMnecessary to give 50% inhibition of ras-luciferase expression). Thelonger phosphorothioates targeted to the codon-12 point mutation, whiledisplaying substantial antisense activity toward ras-luciferaseexpression, did not demonstrate selective inhibition of expression ofthe mutant form of ras-luciferase. Phosphorothioate oligonucleotidestargeted to the codon-12 point mutation that were less than 17nucleotides in length did not show activity to either form ofras-luciferase. These results demonstrate effective antisense activityof phosphorothioate oligonucleotides targeted to ras sequences.

Antisense Oligonucleotides Specifically Hybridizable with the H-ras AUG:

Three 20-base phosphorothioate oligonucleotides, targeted to the H-rasAUG codon, were compared for their ability to inhibit ras-luciferaseexpression in transient transfection assays as described in Examples2-5. These oligonucleotides, ISIS 2502 (SEQ ID NO: 1) , 2503 (SEQ ID NO:2) and 6186 (SEQ ID NO: 7) shown in Table 2, were tested for inhibitionof ras-luciferase expression at a single dose (100 nM) in HeLa cells.All three AUG-targeted oligonucleotides were effective in inhibitingras-luciferase expression. These three phosphorothioate oligonucleotideswere also prepared with a 2'-O-methyl modification on each sugar. The2'-O-methylated version of ISIS 2503 (SEQ ID NO: 2) also inhibitedras-luciferase expression with an IC50 between 200 and 500 nM. SEQ IDNO: 7 as a 2'-O-methyl gave approximately 40% inhibition at the highestdose (500 nM).

                                      TABLE 2    __________________________________________________________________________    Antisense oligonucleotides targeted to mutant H-ras    (Oligonucleotide sequences shown 5' to 3')                                 IC50 SEQ.ID    ISIS#        TARGET SEQUENCE          (nM) NO.    __________________________________________________________________________    2502        AUG    CTTATATTCCGTCATCGCTC                                 750  1    2503        AUG    TCCGTCATCGCTCCTCAGGG                                  50  2    6186        AUG    TATTCCGTCATCGCTCCTCA                                 --   7    2563        CODON 12               CGACG             --   8    2564        CODON 12               CCGACGG           --   9    2565        CODON 12               ACCGACGGC         --   10    2567        CODON 12               CACCGACGGCG       --   11    2568        CODON 12               ACACCGACGGCGC     --   12    2569        CODON 12               CACACCGACGGCGCC   --   13    3426        CODON 12               CCACACCGACGGCGCC  --   14    3427        CODON 12               CACACCGACGGCGCCC  --   15    2570        CODON 12               CCACACCGACGGCGCCC 100  3    3428        CODON 12               CCCACACCGACGGCGCCC                                 --   16    3429        CODON 12               CCACACCGACGGCGCCCA                                 --   17    2571        CODON 12               CCCACACCGACGGCGCCCA                                 250  4    2566        CODON 12               GCCCACACCGACGGCGCCCAC                                 250  5    2560        CODON 12               TGCCCACACCGACGGCGCCCACC                                 750  6    2561        CODON 12               TTGCCCACACCGACGGCGCCCACCA                                 1000 18    2907        CODON 12               CCACACCGCCGGCGCCC --   19        (normal)    __________________________________________________________________________

Oligonucleotide Length Affects Antisense Activity and Specificity:

Oligonucleotides targeted to the H-ras codon- 12 point mutation alsowere effective in inhibiting expression of ras-luciferase. A series ofeleven phosphorothioate oligonucleotides, ranging in length between 5and 25 bases, were made and tested for ability to inhibit mutant andwild type ras-luciferase in transient transfection assays as describedin Examples 2-5. The oligonucleotides are shown in Table 2. At 100 nMoligonucleotide concentration, oligonucleotides 15 bases or greater inlength were found to inhibit expression of the mutant H-ras target.Selective inhibition of mutant over wild type ras-luciferase expressionwas observed for oligonucleotides between 15 and 19 bases in length. Themaximum selectivity observed for inhibition of mutant ras-luciferaserelative to wild type was for the 17-mer 2570 (SEQ ID NO: 3) and wasapproximately 4-fold. In order to demonstrate that 2570 was acting in asequence-specific manner, a variant of this compound was tested (2907;SEQ ID NO: 19) in which the central adenosine residue was replaced withcytosine, making this oligonucleotide perfectly complementary to thenormal H-ras target. Hence, this oligonucleotide will contain a singlemismatch at the center of the oligonucleotide/RNA duplex when fullyhybridized to the mutant H-ras sequence. Oligonucleotide 2907selectively inhibited expression of normal ras-luciferase (88%inhibition) relative to mutant ras-luciferase (5% inhibition).

Two 16-mers and two 18-mers complementary to the mutant codon-12 region(Table 2) were tested as described in Examples 2-5. FIG. 1 shows theresults of an experiment in which antisense activity and mutantselectivity was determined for oligonucleotides of length 13, 15, 16,17, 18 and 19 bases in a dose-dependent manner. The results obtainedwith these oligonucleotides demonstrated that the compounds that wereactive against mutant H-ras sequences also showed selectivity;oligonucleotides of length 16 (SEQ ID NO: 14 and SEQ ID NO: 15) and 17bases (SEQ ID NO: 3) displayed the greatest selectivity (4- and 5-fold,respectively). The 13 base compound, 2568 (SEQ ID NO: 12), did notdisplay antisense activity at any of the tested concentrations.

Chimeric 2'-O-methyl Oligonucleotides with Deoxy Gaps:

Based on the sequence of the mutant-selective 17-mer (2570), a series ofchimeric phosphorothioate 2'-O-methyl oligonucleotides were synthesizedin which the end regions consisted of 2'-O-methyl nucleosides and thecentral residues formed a "deoxy gap". The number of deoxy residuesranged from zero (full 2'-O-methyl) to 17 (full deoxy). Theseoligonucleotides are shown in Table 3.

                  TABLE 3    ______________________________________    Chimeric phosphorothioate oligonucleotides    having 2'-O-methyl ends (bold) and central deoxy gap    (Mutant codon-12 target)    OLIGO #           DEOXY     SEQUENCE         SEQ ID NO    ______________________________________    4122   0         CCACACCGACGGCGCCC                                      3    3975   1         CCACACCGACGGCGCCC                                      3    3979   3         CCACACCGACGGCGCCC                                      3    4236   4         CCACACCGACGGCGCCC                                      3    4242   4         CCACACCGACGGCGCCC                                      3    3980   5         CCACACCGACGGCGCCC                                      3    3985   7         CCACACCGACGGCGCCC                                      3    3984   9         CCACACCGACGGCGCCC                                      3    2570   17        CCACACCGACGGCGCCC                                      3    ______________________________________

These oligonucleotides were characterized for hybridization efficiencyas described in Example 6, ability to direct RNase H cleavage in vitrousing mammalian RNase H as described in Example 8, and for antisenseactivity. Antisense activity against full length H-ras mRNA wasdetermined using a transient co-transfection reporter gene system inwhich H-ras gene expression was monitored using a ras-responsiveenhancer element linked to the reporter gene luciferase, as described inExample 9.

Antisense Activity of Deoxy-gapped Oligonucleotides Against Full Lengthras mRNA:

The beneficial properties of enhanced target affinity conferred by2'-O-methyl modifications can be exploited for antisense inhibitionprovided these compounds are equipped with RNase H-sensitive deoxy gapsof the appropriate length. 2'-O-methyl deoxy gap oligonucleotides weretested for antisense activity against the full length H-ras mRNA usingthe H-ras transactivation reporter gene system described in Example 9.Antisense experiments were performed initially at a singleoligonucleotide concentration (100 nM). Chimeric 2'-O-methyloligonucleotides containing deoxy gaps of five or more residuesinhibited H-ras gene expression. The full deoxy compound gaveapproximately 50% inhibition. The fully 2'-O-methyl, 1-deoxy and 3-deoxygave no inhibition. The 5-deoxy, 7-deoxy and 9-deoxy compounds gaveapproximately 85%, 95% and 90% inhibition, respectively. These compoundsdisplayed activities greater than that of the full deoxy parentcompound.

Dose response experiments were performed using these active compounds,along with the 2'-O-methyl chimeras containing four deoxy residues.Oligonucleotide-mediated inhibition of full-length H-ras by theseoligonucleotides was dose-dependent. The most active compound was theseven-residue deoxy chimera, which displayed an activity approximatelyfive times greater than that of the full deoxy oligonucleotide.

Shortened Chimeric Oligonucleotides:

Enhanced target affinity conferred by the 2'-O-methyl modifications wasfound to confer activity on short chimeric oligonucleotides. A series ofshort 2'-O-methyl chimeric oligonucleotides were tested for T_(m) andantisense activity vs. full length ras as described in Example 9. Table4 shows T_(m) s for oligonucleotides 11, 13, 15 and 17 nucleotides inlength, having deoxy gaps either 5 bases long or 7 bases long. In sharpcontrast to the full deoxy 13-mer, both 2'-O-methyl chimeric 13-mersinhibited ras expression, and one of the 11-mers was also active. Thisis shown in FIG. 2.

                  TABLE 4    ______________________________________    LENGTH T.sub.m (°C.)                    SEQUENCE         SEQ ID NO:    ______________________________________    17     77.2     CCACACCGACGGCGCCC                                     3    15     69.8     CACACCGACGGCGCC  13    13     62.1     ACACCGACGGCGC    12    11     47.3     CACCGACGGCG      11    17     74.6     CCACACCGACGGCGCCC                                     3    15     66.2     CACACCGACGGCGCC  13    13     58.0     ACACCGACGGCGC    12    11     27.7     CACCGACGGCG      11    ______________________________________

Relative antisense activity and ability to activate RNase H cleavage invitro by chimeric 2'-O-methyl oligonucleotides is well correlated withdeoxy length (FIG. 3).

Asymmetrical Deoxy Gaps:

It is not necessary that the deoxy gap be in the center of the chimericmolecule. It was found that chimeric molecules having the nucleotides ofthe region at one end modified at the 2' position to enhance binding andthe remainder of the molecule unmodified (2'deoxy) can still inhibit rasexpression. Oligonucleotides of SEQ ID NO: 3 (17-mer complementary tomutant codon 12) in which a 7-deoxy gap was located at either the 5' or3' side of the 17-mer, or at different sites within the middle of themolecule, all demonstrated RNase H activation and antisense activity.However, a 5-base gap was found to be more sensitive to placement, assome gap positions rendered the duplex a poor activator of RNase H and apoor antisense inhibitor. Therefore, a 7-base deoxy gap is preferred.

Other Sugar Modifications:

The effects of other 2' sugar modifications besides 2'-O-methyl onantisense activity in chimeric oligonucleotides have been examined.These modifications are listed in Table 5, along with the T_(m) valuesobtained when 17-mer oligonucleotides having 2'-modified nucleotidesflanking a 7-base deoxy gap were hybridized with a 25-meroligoribonucleotide complement as described in Example 6. A relationshipwas observed for these oligonucleotides between alkyl length at the 2'position and T_(m). As alkyl length increased, T_(m) decreased. The2'-fluoro chimeric oligonucleotide displayed the highest T_(m) of theseries.

                  TABLE 5    ______________________________________    Correlation of T.sub.m with Antisense Activity    2'-modified 17-mer with 7-deoxy gap    CCACACCGACGGCGCCC (SEQ ID NO: 3)    2' MODIFICATION   T.sub.m (°C.)                              IC50 (nM)    ______________________________________    Deoxy             64.2    150    O-Pentyl          68.5    150    O-Propyl          70.4    70    O-Methyl          74.7    20    Fluoro            76.9    10    ______________________________________

These 2' modified oligonucleotides were tested for antisense activityagainst H-ras using the transactivation reporter gene assay described inExample 9. As shown in Table 5, all of these 2' modified chimericcompounds inhibited ras expression, with the 2'-fluoro 7-deoxy-gapcompound the most active. A 2'-fluoro chimeric oligonucleotide with acentered 5-deoxy gap was also active.

Chimeric phosphorothioate oligonucleotides having SEQ ID NO: 3 having2'-O-propyl regions surrounding a 5-base or 7-base deoxy gap werecompared to 2'-O-methyl chimeric oligonucleotides. ras expression in T24cells was inhibited by both 2'-O-methyl and 2'-O-propyl chimericoligonucleotides with a 7-deoxy gap and a uniform phosphorothioatebackbone. When the deoxy gap was decreased to five nucleotides, only the2'-O-methyl oligonucleotide inhibited ras expression.

Antisense Oligonucleotide Inhibition of H-ras Gene Expression in CancerCells:

Two phosphorothioate oligonucleotides (2502, 2503) complementary to theras AUG region were tested as described in Example 10, along withchimeric oligonucleotides (4998, 5122) having the same sequence and7-base deoxy gaps flanked by 2'-O-methyl regions. These chimericoligonucleotides are shown in Table 6.

                  TABLE 6    ______________________________________    Chimeric phosphorothioate oligonucleotides    having 2'-O-methyl ends (bold) and central deoxy gap    (AUG target)    OLIGO #           DEOXY    SEQUENCE           SEQ ID NO:    ______________________________________    2502   20       CTTATATTCCGTCATCGCTC                                       1    4998   7        CTTATATTCCGTCATCGCTC                                       1    2503   20       TCCGTCATCGCTCCTCAGGG                                       2    5122   7        TCCGTCATCGCTCCTCAGGG                                       2    ______________________________________

Compound 2503 inhibited ras expression in T24 cells by 71%, and thechimeric compound (4998) inhibited ras mRNA even further (84%inhibition). Compound 2502, also complementary to the AUG region,decreased ras RNA levels by 26% and the chimeric version of thisoligonucleotide (5122) demonstrated 15% inhibition. Also included inthis assay were two oligonucleotides targeted to the mutant codon 12.Compound 2570 (SEQ ID NO: 3) decreased ras RNA by 82% and the 2'-Omethylchimeric version of this oligonucleotide with a seven-deoxy gap (3985)decreased ras RNA by 95%.

Oligonucleotides 2570 and 2503 were also tested to determine theireffects on ras expression in HeLa cells, which have a wild-type (i.e.,not activated) H-ras codon 12. While both of these oligonucleotidesinhibited ras expression in T24 cells (having activated codon 12), onlythe oligonucleotide (2503) specifically hybridizable with the ras AUGinhibited ras expression in HeLa cells. Oligonucleotide 2570 (SEQ ID NO:3), specifically hybridizable with the activated codon 12, did notinhibit ras expression in HeLa cells, because these cells lack theactivated codon-12 target.

Oligonucleotide 2570, a 17-mer phosphorothioate oligonucleotidecomplementary to the codon 12 region of activated H-ras, was tested forinhibition of ras expression (as described in Example 10) in T24 cellsalong with chimeric phosphorothioate 2'-O-methyl oligonucleotides 3980,3985 and 3984, which have the same sequence as 2570 and have deoxy gapsof 5, 7 and 9 bases, respectively (shown in Table 3). The fully 2'-deoxyoligonucleotide 2570 and the three chimeric oligonucleotides decreasedras mRNA levels in T24 cells. Compounds 3985 (7-deoxy gap) and 3984(9-deoxy gap) decreased ras mRNA by 81%; compound 3980 (5-deoxy gap)decreased ras mRNA by 61%. Chimeric oligonucleotides having thissequence, but having 2'-fluoro-modified nucleotides flanking a 5-deoxy(4689) or 7-deoxy (4690) gap, inhibited ras mRNA expression in T24cells, with the 7-deoxy gap being preferred (82% inhibition, vs 63%inhibition for the 2'-fluoro chimera with a 5-deoxy gap).

Antisense Oligonucleotide Inhibition of Proliferation of Cancer Cells:

Three 17-mer oligonucleotides having the same sequence (SEQ ID NO: 3),complementary to the codon 12 region of activated ras, were tested foreffects on T24 cancer cell proliferation as described in Example 11.3985 has a 7-deoxy gap flanked by 2'-O-methyl nucleotides, and 4690 hasa 7-deoxy gap flanked by 2'-F nucleotides (all are phosphorothioates).Effects of these oligonucleotides on cancer cell proliferationcorrelated well with their effects on ras mRNA expression shown byNorthern blot analysis: oligonucleotide 2570 inhibited cellproliferation by 61%, the 2'-O-methyl chimeric oligonucleotide 3985inhibited cell proliferation by 82%, and the 2'-fluoro chimeric analoginhibited cell proliferation by 93%.

In dose-response studies of these oligonucleotides on cellproliferation, the inhibition was shown to be dosedependent in the 25nM-100 nM range. IC50 values of 44 nM, 61 nM and 98 nM could be assignedto oligonucleotides 4690, 3985 and 2570, respectively. The randomoligonucleotide control had no effect at the doses tested.

The effect of ISIS 2570 on cell proliferation was cell type-specific.The inhibition of T24 cell proliferation by this oligonucleotide wasfour times as severe as the inhibition of HeLa cells by the sameoligonucleotide (100 nM oligonucleotide concentration). ISIS 2570 istargeted to the activated (mutant) ras codon 12, which is present in T24but lacking in HeLa cells, which have the wild-type codon 12.

Chimeric Backbone-modified Oligonucleotides:

Oligonucleotides discussed in previous examples have had uniformphosphorothioate backbones. The 2'modified chimeric oligonucleotidesdiscussed above are not active in uniform phosphodiester backbones. Achimeric oligonucleotide was synthesized (ISIS 4226) having 2'-O-methylregions flanking a 5-nucleotide deoxy gap, with the gap region having aP═S backbone and the flanking regions having a P=O backbone. Anotherchimeric oligonucleotide (ISIS 4223) having a P═O backbone in the gapand P═S in flanking regions was also made. These oligonucleotides areshown in Table 7.

Additional oligonucleotides were synthesized, completely 2' deoxy andhaving phosphorothioate backbones containing either a singlephosphodiester (ISIS 4248), two phosphodiesters (ISIS 4546), threephosphodiesters (ISIS 4551), four phosphodiesters (ISIS 4593), fivephosphodiesters (ISIS 4606) or ten phosphodiester linkages (ISIS-4241)in the center of the molecule. These oligonucleotides are also shown inTable 7.

                  TABLE 7    ______________________________________    Chimeric backbone (P = S/P = 0) oligonucleotides    having 2'-O-methyl ends (bold) and central deoxy gap    (backbone linkages indicated by s (P = S) or o (P = O)    Mutant codon-12 target                                          SEQ                                          ID    OLIGO #           P = S  SEQUENCE                NO:    ______________________________________    2570   16     CsCsAsCsAsCsCsGsAsCsGsGsCsGsCsCsC                                          3    4226   5      CoCoAoCoAoCsCsGsAsCsGoGoCoGoCoCoC                                          3    4233   11     CsCsAsCsAsCoCoGoAoCoGsGsCsGsCsCsC                                          3    4248   15     CsCsAsCsAsCsCsGsAoCsGsGsCsGsCsCsC                                          3    4546   14     CsCsAsCsAsCsCsGoAoCsGsGsCsGsCsCsC                                          3    4551   13     CsCsAsCsAsCsCsGoAoCoGsGsCsGsCsCsC                                          3    4593   12     CsCsAsCsAsCsCoGoAoCoGsGsCsGsCsCsC                                          3    4606   11     CsCsAsCsAsCsCoGoAoCoGoGsCsGsCsCsC                                          3    4241   6      CsCsAsCoAoCoCoGoAoCoGoGoCoGsCsCsC                                          3    ______________________________________

Oligonucleotides were incubated in crude HeLa cellular extracts at 37°C. to determine their sensitivity to nuclease degradation as describedin Dignam et al., Nucleic Acids Res. 1983, 11, 1475-1489. Theoligonucleotide (4233) with a five-diester gap betweenphosphorothioate/2'-O-methyl wings had a T_(1/2) of 7 hr. Theoligonucleotide with a five-phosphorothioate gap in aphosphorothioate/2'-O-methyl molecule had a T_(1/2) of 30 hours. In theset of oligonucleotides having one to ten diester linkages, theoligonucleotide (4248) with a single phosphodiester linkage was asstable to nucleases as was the full-phosphorothioate molecule, ISIS2570, showing no degradation after 5 hours in HeLa cell extract.Oligonucleotides with two-, three- and four-diester gaps had T_(1/2) ofapproximately 5.5 hours, 3.75 hours, and 3.2 hours, and oligonucleotideswith five or ten deoxy linkages had T_(1/2) of 1.75 hours and 0.9 hours,respectively.

Antisense Activity of Chimeric Backbone-modified Oligonucleotides:

A uniform phosphorothioate backbone is not required for antisenseactivity. ISIS 422G and ISIS 4233 were tested in the ras-luciferasereporter system for effect on ras expression as described in Examples2-5, along with ISIS 2570 (fully phosphorothioate/all deoxy), ISIS 3980(fully phosphorothioate, 2'-O-methyl wings with deoxy gap) and ISIS 3961(fully phosphodiester, 2'-O-methyl wings with deoxy gap). All of theoligonucleotides having a P═S (i.e., nuclease-resistant) gap regioninhibited ras expression. The two completely 2'deoxy oligonucleotideshaving phosphorothioate backbones containing either a singlephosphodiester (ISIS 4248) or ten phosphodiester linkages (ISIS 4241) inthe center of the molecule were also assayed for activity. The compoundcontaining a single P═O was just as active as a full P═S molecule, whilethe same compound containing ten P═O was completely inactive.

Chimeric phosphorothioate oligonucleotides of SEQ ID NO: 3 were made,having a phosphorothioate backbone in the 7-base deoxy gap region only,and phosphodiester in the flanking regions, which were either2'-O-methyl or 2'-O-propyl. The oligonucleotide with the 2'-O-propyldiester flanking regions was able to inhibit ras expression.

Inhibition of Ras-luciferase Gene Expression by AntisenseOligonucleotides Containing Modified Bases:

A series of antisense phosphorothioate oligonucleotides complementary tothe codon-12 point mutation of activated ras were synthesized asdescribed, having a 2-(amino)adenine at the position complementary tothe uracil of the mutated codon 12. Because the amino group at the2-position of the adenine is able to hydrogen bond with the oxygen inthe 2-position on the uracil, three hydrogen bonds instead of the usualtwo are formed. This serves to greatly stabilize the hybridization ofthe 2-(amino)adenine-modified antisense oligonucleotide to the activatedras gene, while destabilizing or having no net effect on the stabilityof this oligonucleotide to the wild-type codon 12, because of themodified A-G mismatch at this position. This increases the specificityof the modified oligonucleotide for the desired target.

An oligonucleotide having a single 2,6- (diamino) adenosine at thisposition in an otherwise unmodified uniform phosphorothioate 17-mer(sequence identical to 2570, SEQ ID NO: 3) was found to be at least aseffective an RNase H substrate as the 2570 sequence. It is thereforeexpected to be an effective antisense molecule. An oligonucleotidehaving a single 2, - (diamino) adenosine at this position in a deoxygapped phosphorothioate oligonucleotide of the same sequence alsodemonstrates RNase H activation.

In Vivo Anti-tumor Data:

ISIS 2503 (SEQ ID NO: 2) has been evaluated for activity against humantumors in vivo as described in Examples 13 and 14. These studiesemployed a human lung adenocarcinoma cell line (A549) which wassubcutaneously implanted into nude mice, resulting in tumor growth atsite of implantation. Since these cells do not contain a mutation in theH-ras gene, but do express normal H-ras, only the AUG-directedoligonucleotide ISIS 2503 was evaluated for anti-tumor activity.

In the first study, phosphorothioate oligonucleotides in saline wereadministered by intraperitoneal injection at a dosage of 20 mg/kg. Drugtreatment was initiated at the time tumors first became visible (28 daysfollowing tumor cell inoculation) and treatments were performed everyother day. As shown in FIG. 4, no effect on tumor growth was observedafter treatment with the unrelated control phosphorothioateoligonucleotide ISIS 1082 (SEQ ID NO: 55). However, significantinhibition of tumor growth was observed for the H-ras-specificoligonucleotide ISIS 2503 (SEQ ID NO: 2). The anti-tumor effects of theH-ras compound were first observed 20 days following initiation of drugtreatment and continued throughout the duration of the study.

In a second study, phosphorothioate oligonucleotides were prepared in acationic lipid formulation (DMRIE:DOPE) and administered by subcutaneousinjection as described in Example 15. Drug treatment was initiated oneweek following tumor cell inoculation and was performed three times aweek for only four weeks. As was observed in the first study,administration of the H-ras-specific compound ISIS 2503 (SEQ ID NO: 2)caused a marked reduction in tumor growth whereas the unrelated controloligonucleotide (ISIS 1082) had no significant effect (FIG. 5).Reduction in tumor volume was first observed 20 days followingappearance of visible tumors and continued over time throughout theremainder of the study.

Stability of 2'-modified Phosphodiester Oligonucleotides in Cells:

Modification of oligonucleotides to confer nuclease stability isrequired for antisense activity in cells. Certain modifications at the2' position of the sugar have been found to confer nuclease resistancesufficient to elicit antisense effects in cells without any backbonemodification. A uniformly 2'-propoxy modified phosphodiesteroligonucleotide (SEQ ID NO: 3) was found to inhibit H-ras expression inT24 cells, 24 hours after administration, at a level equivalent to aphosphorothioate 2'-deoxyoligonucleotide having the same sequence.Uniform 2'-methoxy phosphodiester oligonucleotide also showed someactivity. 2'-pentoxy modifications were found to be at least as activeas the 2'-propoxy.

Antisense Oligonucleotides Active Against Ki-ras:

Oligonucleotides were designed to be complementary to the5'-untranslated region, 3'-untranslated region and coding region of thehuman Ki-ras oncogene. McGrath, J. P. et al. Nature 1983, 304, 501-506.Of the latter, oligonucleotides were targeted to codons 12 and 61 whichare known sites of mutation that lead to Ki-ras-mediated transformation,and also to codon 38, which is not known to be involved intransformation. The oligonucleotides are shown in Table 8.

                  TABLE 8    ______________________________________    Antisense Oligonucleotides Complementary to Human Ki-ras    ISIS                                 SEQ ID    #    SEQUENCE             TARGET     NO:    ______________________________________    6958 CTG CCT CCG CCG CCG CGG CC                              5' UTR/5'  20                              cap    6957 CAG TGC CTG CGC CGC GCT CG                              5'-UTR     21    6956 AGG CCT CTC TCC CGC ACC TG                              5'-UTR     22    6953 TTC AGT CAT TTT CAG CAG GC                              AUG        23    6952 TTA TAT TCA GTC ATT TTC AG                              AUG        24    6951 CAA GTT TAT ATT CAG TCA TT                              AUG        25    6950 GCC TAC GCC ACC AGC TCC AAC                              Codon 12   26                              (WT)    6949 CTA CGC CAC CAG CTC CA                              Codon 12   27                              (WT)    6948 G TAC TCC TCT TGA CCT GCT                              Codon 61   28         GT                   (WT)    6947 CCT GTA GGA ATC CTC TAT TGT                              Codon 38   29    6946 GGT AAT GCT AAA ACA AAT GC                              3'-UTR     30    6945 GGA ATA CTG GCA CTT CGA GG                              3'-UTR     31    7453 TAC GCC AAC AGC TCC  Codon 12   32                              (G→T mut.)    7679 TTT TCA GCA GGC CTC TCT CC                              5'-UTR/AUG 33    ______________________________________

Twelve Ki-ras-specific oligonucleotides were screened for antisenseactivity against three colon carcinoma cell lines that contain amutation at codon 12 in the Ki-ras oncogene and evaluated by measurementof Ki-ras mRNA levels. As shown in FIG. 6, half of the tested compoundsdisplayed significant activity (at least 40% inhibition) against theKi-ras transcript, with the most active compounds being targeted to the5'- and 3'-untranslated regions. However, significant inhibition ofKi-ras expression was also observed for compounds directed against wildtype codons 12 and 61. Compounds that displayed significant activitywere effective against all three carcinoma cell lines tested.

Dose response analysis of these compounds demonstrated that ISIS 6958and ISIS 6957, both of which target the 5'-UTR, are the most potentinhibitors of Ki-ras in this series of oligonucleotides. Theseoligonucleotides were examined for their ability to inhibitproliferation of Ki-ras transformed cell lines. The colon carcinoma cellline SW480 was treated with a single dose of oligonucleotide (200 nM)and cell number was determined over a five-day period. As shown in FIG.7 both Ki-ras specific oligonucleotides were effective inhibitors ofproliferation of SW480 cells, with ISIS 6957 (SEQ ID NO: 21) showinggreater activity than ISIS 6958 (SEQ ID NO: 20). This difference inactivity correlates well with the inhibition of Ki-ras mRNA expression(FIG. 6).

Selectivity of Inhibition of Mutant Ki-ras Relative to Normal Ki-ras:

Oligonucleotides targeted to Ki-ras have been examined for their abilityto selectively inhibit mutant Ki-ras relative to normal Ki-ras. Two celllines were employed: the SW480 cell line that expresses mutant Ki-ras(codon 12, G to T transversion) and a cell line (HeLa) that expressesnormal Ki-ras. Two oligonucleotides were tested: ISIS 6957, SEQ ID NO:21, a 20 mer phosphorothioate targeted to the 5'-untranslated region ofKi-ras, and ISIS 7453, SEQ ID NO: 32, a 15 mer phosphorothioate targetedto the Ki-ras codon 12 region. Ki-ras mRNA levels were measured 24 hoursafter treatment. The codon 12-directed compound was effective in thecell line expressing mutant Ki-ras (87% inhibition vs. 18% inhibition inHeLa cells). However, the Ki-ras oligonucleotide targeted to the5'-untranslated region was a potent inhibitor (95% inhibition) of Ki-rasexpression in both cell lines. Selectivity for mutant Ki-ras was foundto be dependent on oligonucleotide concentration and affinity for theRNA target.

Ki-ras Oligonucleotides with Deoxy Gaps:

Phosphorothioate oligonucleotides (SEQ ID NO: 21, targeted to the5'-untranslated region of Ki-ras) were synthesized with 2'-O-methylmodifications flanking central 2'-deoxy gap regions of 6 or 8nucleotides in length. Both gapped oligonucleotides were active againstKi-ras expression as determined by Northern blot analysis. A uniformly2'-O-methylated compound (no deoxy gap) was inactive. An additionaloligonucleotide, ISIS 7679 (SEQ ID NO: 33, complementary to the 5'untranslated/AUG region of Ki-ras), was also found to be active whensynthesized with a 6- or 8- nucleotide deoxy gap.

2'-Methoxyethoxy Analogs of ISIS 2503 (H-ras):

A series of chimeric oligonucleotides were synthesized with the ISIS2503 sequence (SEQ ID NO: 2) and various arrangements of2'-methoxyethoxy (2'-MOE) modifications. These are shown in Table 9. Allbackbone linkages are phosphorothioates.

                  TABLE 9    ______________________________________    2'-MOE analogs of ISIS 2503    Positions with 2'-MOE are shown in bold    ISIS #   Sequence (5'- -3')  SEQ ID NO:    ______________________________________    13905    TCCGTCATCGCTCCTCAGGG                                 2    13907    TCCGTCATCGCTCCTCAGGG                                 2    13909    TCCGTCATCGCTCCTCAGGG                                 2    13911    TCCGTCATCGCTCCTCAGGG                                 2    13917    TCCGTCATCGCTCCTCAGGG                                 2    13919    TCCGTCATCGCTCCTCAGGG                                 2    13920    TCCGTCATCGCTCCTCAGGG                                 2    13923    TCCGTCATCGCTCCTCAGGG                                 2    13926    TCCGTCATCGCTCCTCAGGG                                 2    13927    TCCGTCATCGCTCCTCAGGG                                 2    ______________________________________

These oligonucleotides (except for 13919 and 13927 which have not yetbeen tested) were tested for the ability to reduce H-ras mRNA levels inT24 cells as described in Example 10 except that oligonucleotide andlipofectin were mixed in OptiMEM and kept at a constant ratio of 2.5ug/ml lipofectin per 100 nM oligonucleotide. All of the tested compoundshad activity comparable to ISIS 2503, the parent compound, with IC50'sof 50 nM or below. For this reason oligonucleotides containing one ormore 2'-MOE modifications are preferred for reducing ras expression.Dose responses for these compounds are shown in FIG. 8. ISIS 13177(TCAGTAATAGCCCCACATGG; SEQ ID NO: 34) is a phosphorothioateoligodeoxynucleotide scrambled control for SEQ ID NO: 2.

MMI Analogs of ISIS 2503 (H-ras):

A series of chimeric oligonucleotides were synthesized with the ISIS2503 sequence (SEQ ID NO: 2) and various placements ofmethylene(methylimino)backbone linkages. These are shown in Table 10.For ease of synthesis, dimers incorporating an MMI linkage were used inmaking these oligonucleotides. Dimers containing MMI backbone linkagesare indicated by bold lettering. "o"indicates a phosphodiester linkagebetween MMI dimers. "s"indicates a phosphorothioate linkage between MMIdimers. All unmarked linkages are phosphorothioates.

                  TABLE 10    ______________________________________    MMI analogs of ISIS 2503    ISIS #   Sequence (5'- -3')  SEQ ID NO:    ______________________________________    14896    TCCGTCATCGCTCCTCAGGG                                 2    14897    TC.sub.o CGTCATCGCTCCTCAG.sub.o GG                                 2    14898    TC.sub.s CGTCATCGCTCCTCAG.sub.s GG                                 2    14899    TC.sub.o CG.sub.o TCATCGCTCCTC.sub.o A.sub.o GGG                                 2    14900    TC.sub.s CG.sub.s TCATCGCTCCTC.sub.s AG.sub.s AG                                 2    ______________________________________

These compounds were tested for their ability to reduce H-ras mRNAlevels in T24 cells as described in Example 10 except thatoligonucleotide and lipofectin were mixed in OptiMEM and kept at aconstant ratio of 2.5 μg/ml lipofectin per 100 nM oligonucleotide. Asshown in FIG. 9, all of these compounds were able to reduce mRNA levelsby 80% or more at doses of 500 nM and below. ISIS 13177 (SEQ ID NO: 34)is a phosphorothioate oligodeoxynucleotide scrambled control for SEQ IDNO: 2. With the exception of ISIS 14899, all the MMI compounds were moreactive than the parent deoxyphosphorothioate compound, ISIS 2503.Several compounds (ISIS 14896, 14897, 14898) achieved nearly completeablation of ras mRNA. Oligonucleotides containing one or more MMImodifications are therefore highly preferred for reducing rasexpression.

Antisense Oligonucleotides Active Against N-ras:

A series of phosphorothioate oligodeoxynucleotides were designed totarget human N-ras using the published sequence (Genbank accessionnumber HSNRASR, x02751). These compounds were tested for their abilityto reduce N-ras levels in T24 cells as described in Example 10 exceptthat the probe was an N-ras cDNA probe (purchased from Oncogene Science,Cambridge Mass.; catalog no. HP129) and oligonucleotide and lipofectinwere mixed in OptiMEM and kept at a constant ratio of 2.5 ug/mllipofectin per 100 nM oligonucleotide.

These oligonucleotides, and the percent reduction in N-ras mRNAdemonstrated for each, are shown in Table 11. Oligonucleotides shown inbold (SEQ ID NO: 44, 45, 46, 47, 49 and 52) demonstrated greater than30% reduction of ras mRNA when screened at a 300 nM dose and areconsidered active in this assay. These sequences are thereforepreferred. Of these oligonucleotides 14686, 14687, 14688, 14691 and14694 (SEQ ID NO: 44, 45, 46, 49 and 52) showed greater than 50%inhibition. Dose response curves were obtained for oligonucleotides14677, 14678, 14686, 14687, 14688, 14689, 14690, 14891, 14692, 14693,and 14694. These are shown in FIG. 10. As can be seen from the figure,ISIS 14686 and ISIS 14691 (SEQ ID NO: 44 and 49, respectively) gavenearly complete ablation of N-ras mRNA at a 400 nM dose.

                  TABLE 11    ______________________________________    Oligonucleotides targeted to human N-ras                                            SEQ    ISIS                     Target  %      ID    #     Sequence (5'- -3') Region  Reduced                                            NO:    ______________________________________    14677 CCGGGTCCTAGAAGCTGCAG                             5' UTR  0.0    35    14678 TAAATCAGTAAAAGAAACCG                             5' UTR  0.0    36    14679 GGACACAGTAACCAGGCGGC                             5' UTR  0.0    37    14680 AACAGAAGCTACACCAAGGG                             5' UTR  0.0    38    14681 CAGACCCATCCATTCCCGTG                             5' UTR  0.0    39    14682 GCCAAGAAATCAGACCCATC                             5' UTR  0.0    40    14683 AGGGGGAAGATAAAACCGCC                             5' UTR  0.0    41    14684 CGCTTCCATTCTTTCGCCAT                             5' UTR  0.0    42    14685 CCGCACCCAGACCCGCCCCT                             5' UTR  0.0    43    14686 CAGCCCCCACCAAGGAGCGG                             5' UTR  61.0   44    14687 GTCATTTCACACCAGCAAGA                             AUG     50.2   45    14688 CAGTCATTTCACACCAGCAA                             AUG     60.5   46    14689 CTCAGTCATTTCACACCAGC                             AUG     38.4   47    14690 CGTGGGCTTGTTTTGTATCA                             Coding  0.2    48    14691 CCATACAACCCTGAGTCCCA                             3' UTR  58.3   49    14692 CAGACAGCCAAGTGAGGAGG                             3' UTR  0.0    50    14693 CCAGGGCAGAAAAATAACAG                             3' UTR  0.0    51    14694 TTTGTGCTGTGGAAGAACCC                             3' UTR  50.7   52    14695 GCTATTAAATAACAATGCAC                             3' UTR  0.0    53    14696 ACTGATCACAGCTATTAAAT                             3' UTR  0.0    54    ______________________________________

The following examples illustrate the present invention and are notintended to limit the same.

EXAMPLES EXAMPLE 1 Synthesis and Characterization of Oligonucleotides

Unmodified oligodeoxynucleotides are synthesized on an automated DNAsynthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl-phosphoramidites are purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2M solution of ³H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step.

2'-methoxy oligonucleotides were synthesized using 2'-methoxyβ-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham, Mass.) andthe standard cycle for unmodified oligonucleotides, except the wait stepafter pulse delivery of tetrazole and base was increased to 360 seconds.Other 2'-alkoxy oligonucleotides were synthesized by a modification ofthis method, using appropriate 2'-modified amidites such as thoseavailable from Glen Research, Inc., Sterling, Va.

2'-fluoro oligonucleotides were synthesized as described in Kawasaki etal., J. Med. Chem. 1993, 36, 831-841. Briefly, the protected nucleosideN⁶ -benzoyl-2'-deoxy-2'-fluoroadenosine was synthesized utilizingcommercially available 9-β-D-arabinofuranosyladenine as startingmaterial and by modifying literature procedures whereby the 2'-α-fluoroatom is introduced by a S_(N) 2-displacement of a 2'-β-O-trifyl group.Thus N⁶ -benzoyl-9-β-D-arabinofuranosyladenine was selectively protectedin moderate yield as the 3',5'-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N⁶ -benzoyl groups was accomplished usingstandard methodologies and standard methods were used to obtain the5'-dimethoxytrityl(DMT) and 5'-DMT-3'-phosphoramidite intermediates.

The synthesis of 2'-deoxy-2'-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected 9-β-D-arabinofuranosylguanineas starting material, and conversion to the intermediatediisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS groupwas followed by protection of the hydroxyl group with THP to givediisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5'-DMT- and5'-DMT-3'-phosphoramidites.

Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by themodification of a known procedure in which 2,2'-anhydro-1-β-D-arabinofuranosyluracil was treated with 70% hydrogenfluoride-pyridine. Standard procedures were used to obtain the 5'-DMTand 5'-DMT-3'phosphoramidites.

2'-deoxy-2'-fluorocytidine was synthesized via amination of2'-deoxy-2'-fluorouridine, followed by selective protection to give N⁴-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures were used toobtain the 5'-DMT and 5'-DMT-3'phosphoramidites.

2'-(2-methoxyethyl)-modified amidites are synthesized according toMartin, P., Helv. Chim. Acta 1995, 78,486-504. For ease of synthesis,the last nucleotide was a deoxynucleotide. 2'--O--CH₂ CH₂ OCH₃ cytosinesmay be 5-methyl cytosines.

Synthesis of 5-Methyl Cytosine Monomers 2,2'-Anhydro1-(β-D-arabinofuranosyl)-5-methyluridine!

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279M), diphenyl-carbonate (90.0 g, 0.420M) andsodium bicarbonate (2.0 g, 0.024M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid which was crushed to a light tan powder (57 g, 85%crude yield). The material was used as is for further reactions.

2'-O-Methoxyethyl-5-methyluridine

2,2'-Anhydro-5-methyluridine (195 g, 0.81M), tris(2-methoxyethyl)borate(231 g, 0.98M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155°-160° C., the vessel wasopened and the solution evaporated to dryness and triturated with MeOH(200 mL). The residue was suspended in hot acetone (1 L). The insolublesalts were filtered, washed with acetone (150 mL) and the filtrateevaporated. The residue (280 g) was dissolved in CH₃ CN (600 mL) andevaporated. A silica gel column (3 kg) was packed in CH₂ Cl₂/acetone/MeOH (20:5:3) containing 0.5 Et₃ NH. The residue was dissolvedin CH₂ Cl₂ (250 mL) and adsorbed onto silica (150 g) prior to loadingonto the column. The product was eluted with the packing solvent to give160 g (63%) of product.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine

2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506M) was co-evaporated withpyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). Afirst aliquot of dimethoxytrityl chloride (94.3 g, 0.278M) was added andthe mixture stirred at room temperature for one hour. A second aliquotof dimethoxytrityl chloride (94.3 g, 0.278M) was added and the reactionstirred for an additional one hour. Methanol (170 mL) was then added tostop the reaction. HPLC showed the presence of approximately 70%product. The solvent was evaporated and triturated with CH₃ CN (200 mL).The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500 mLof saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂ SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃ NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106 g, 0.167M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by tlc by first quenching the tlc sample with the addition ofMeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHC1₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%).

3'-O-Acetyl-2'-O-methoxvethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine:

A first solution was prepared by dissolving3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (96g, 0.144M) in CH₃ CN (700 mL) and set aside. Triethylamine (189 mL,1.44M) was added to a solution of triazole (90 g, 1.3M) in CH₃ CN (1 L),cooled to -5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0°-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the later solution. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1 L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine

A solution of3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141M) in dioxane (500 mL) and NH₄ OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N⁴ -Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165M) wasadded with stirring. After stirring for 3 hours, tlc showed the reactionto be approximately 95% complete. The solvent was evaporated and theresidue azeotroped with MeOH (200 mL) The residue was dissolved in CHCl₃(700 mL) and extracted with saturated NaHCO₃ (2×300 mL) and saturatedNaCl (2×300 mL), dried over MgSO₄ and evaporated to give a residue (96g) . The residue was chromatographed on a 1.5 kg silica column usingEtOAc/Hexane (1:1) containing 0.5% Et₃ NH as the eluting solvent. Thepure product fractions were evaporated to give 90 g (90%) of the titlecompound.

N⁴-Benzoyl-2'-O-methoxvethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-amidit

N⁴ -Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (74g, 0.10M) was dissolved in CH₂ Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tlc showed thereaction to be 95% complete) . The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂ Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

5-methyl-2'-deoxycytidine (5-me-C) containing oligonucleotides weresynthesized according to published methods (Sanghvi et al. Nucl. AcidsRes. 1993, 21, 3197-3203) using commercially available phosphoramidites(Glen Research, Sterling Va. or ChemGenes, Needham Mass.).

Oligonucleotides having methylene(methylimino) (MMI) backbones aresynthesized according to U.S. Pat. No. 5,378,825, which is coassigned tothe assignee of the present invention and is incorporated herein in itsentirety. For ease of synthesis, various nucleoside dimers containingMMI linkages were synthesized and incorporated into oligonucleotides.Other nitrogen-containing backbones are synthesized according to WO92/20823 which is also coassigned to the assignee of the presentinvention and incorporated herein in its entirety.

Oligonucleotides having amide backbones are synthesized according to DeMesmaeker et al. Acc. Chem. Res. 1995, 28, 366-374. The amide moiety isreadily accessible by simple and well-known synthetic methods and iscompatible with the conditions required for solid phase synthesis ofoligonucleotides.

Oligonucleotides with morpholino backbones are synthesized according toU.S. Pat. No. 5,034,506 (Summerton and Weller).

Peptide-nucleic acid (PNA) oligomers are synthesized according to P.E.Nielsen et al. Science 1991, 254, 1497).

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides are purified by precipitation twiceout of 0.5M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotideswere analyzed by polyacrylamide gel electrophoresis on denaturing gelsand judged to be at least 85% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in synthesiswere periodically checked by ³¹ P nuclear magnetic resonancespectroscopy, and for some studies oligonucleotides were purified byHPLC, as described by Chiang et al. J. Biol. Chem. 1991,266:18162-18171. Results obtained with HPLC-purified material weresimilar to those obtained with non-HPLC purified material.

EXAMPLE 2 ras-Luciferase Reporter Gene Assembly

The ras-luciferase reporter genes described in this study were assembledusing PCR technology. oligonucleotide primers were synthesized for useas primers for PCR cloning of the 5'-regions of exon 1 of both themutant (codon 12) and non-mutant (wild-type) human H-ras genes. Theplasmids pT24-C3, containing the c-H-rasl activated oncogene (codon 12,GGC→GTC), and pbc-N1, containing the c-H-ras proto-oncogene, wereobtained from the American Type Culture Collection (Bethesda, Md.). Theplasmid pT3/T7 luc, containing the 1.9 kb firefly luciferase gene, wasobtained from Clontech Laboratories (Palo Alto, Calif.). Theoligonucleotide PCR primers were used in standard PCR reactions usingmutant and non-mutant H-ras genes as templates. These primers produce aDNA product of 145 base pairs corresponding to sequences -53 to +65(relative to the translational initiation site) of normal and mutantH-ras, flanked by NheI and HindIII restriction endonuclease sites. ThePCR product was gel purified, precipitated, washed and resuspended inwater using standard procedures.

PCR primers for the cloning of the P. pyralis (firefly) luciferase genewere designed such that the PCR product would code for the full-lengthluciferase protein with the exception of the amino-terminal methionineresidue, which would be replaced with two amino acids, an amino-terminallysine residue followed by a leucine residue. The oligonucleotide PCRprimers used for the cloning of the luciferase gene were used instandard PCR reactions using a commercially available plasmid(pT3/T7-Luc) (Clontech), containing the luciferase reporter gene, as atemplate. These primers yield a product of approximately 1.9 kbcorresponding to the luciferase gene, flanked by unique HindIII andBssHII restriction endonuclease sites. This fragment was gel purified,precipitated, washed and resuspended in water using standard procedures.

To complete the assembly of the ras-luciferase fusion reporter gene, theras and luciferase PCR products were digested with the appropriaterestriction endonucleases and cloned by three-part ligation into anexpression vector containing the steroid-inducible mouse mammary tumorvirus promotor MMTV using the restriction endonucleases NheI, HindIIIand BssHII. The resulting clone results in the insertion of H-rassequences (-53 to +65) fused in frame with the firefly luciferase gene.The resulting expression vector encodes a ras-luciferase fusion productwhich is expressed under control of the steroid-inducible MMTV promoter.These plasmid constructions contain sequences encoding amino acids 1-22of activated (RA2) or normal (RA4) H-ras proteins fused in frame withsequences coding for firefly luciferase. Translation initiation of theras-luciferase fusion mRNA is dependent upon the natural H-ras AUGcodon. Both mutant and normal H-ras luciferase fusion constructions wereconfirmed by DNA sequence analysis using standard procedures.

EXAMPLE 3 Transfection of Cells with Plasmid DNA

Transfections were performed as described by Greenberg, M. E., inCurrent Protocols in Molecular Biology, (F. M. Ausubel, R. Brent, R. E.Kingston, D. D. Moore, J. A. Smith, J. G. Seidman and K. Strahl, eds.),John Wiley and Sons, NY, with the following modifications. HeLa cellswere plated on 60 mm dishes at 5×10⁵ cells/dish. A total of 10 μg or 12μg of DNA was added to each dish, of which 1 μg was a vector expressingthe rat glucocorticoid receptor under control of the constitutive Roussarcoma virus (RSV) promoter and the remainder was ras-luciferasereporter plasmid. Calcium phosphate-DNA coprecipitates were removedafter 16-20 hours by washing with Tris-buffered saline 50 Mm Tris-Cl (pH7.5), 150 mM NaCl! containing 3 mM EGTA. Fresh medium supplemented with10% fetal bovine serum was then added to the cells. At this time, cellswere pre-treated with antisense oligonucleotides prior to activation ofreporter gene expression by dexamethasone.

EXAMPLE 4 Oligonucleotide Treatment of Cells

Following plasmid transfection, cells were washed with phosphatebuffered saline prewarmed to 37° C. and Opti-MEM containing 5 μg/mL N-1-(2,3-dioleyloxy)propyl!-N,N,N,-trimethylammonium chloride (DOTMA) wasadded to each plate (1.0 ml per well). Oligonucleotides were added from50 μM stocks to each plate and incubated for 4 hours at 37° C. Mediumwas removed and replaced with DMEM containing 10% fetal bovine serum andthe appropriate oligonucleotide at the indicated concentrations andcells were incubated for an additional 2 hours at 37° C. before reportergene expression was activated by treatment of cells with dexamethasoneto a final concentration of 0.2 μM. Cells were harvested and assayed forluciferase activity fifteen hours following dexamethasone stimulation.

EXAMPLE 5 Luciferase Assays

Luciferase was extracted from cells by lysis with the detergent TritonX-100 as described by Greenberg, M. E., in Current Protocols inMolecular Biology, (F. M. Ausubel, R. Brent, R. E. Kingston, D. D.Moore, J. A. Smith, J. G. Seidman and K. Strahl, eds.), John Wiley andSons, NY. A Dynatech ML1000 luminometer was used to measure peakluminescence upon addition of luciferin (Sigma) to 625 μM. For eachextract, luciferase assays were performed multiple times, usingdiffering amounts of extract to ensure that the data were gathered inthe linear range of the assay.

EXAMPLE 6 Melting Curves

Absorbance vs temperature curves were measured at 260 nm using a Gilford260 spectrophotometer interfaced to an IBM PC computer and a GilfordResponse II spectrophotometer. The buffer contained 100 mM Na⁺, 10 mMphosphate and 0.1 mM EDTA, pH 7. Oligonucleotide concentration was 4 μMeach strand determined from the absorbance at 85° C. and extinctioncoefficients calculated according to Puglisi and Tinoco, Methods inEnzymol. 1989, 180, 304-325. T_(m) values, free energies of duplexformation and association constants were obtained from fits of data to atwo state model with linear sloping baselines. Petersheim, M. andTurner, D. H., Biochemistry 1983, 22, 256-263. Reported parameters areaverages of at least three experiments. For some oligonucleotides, freeenergies of duplex formation were also obtained from plots of T_(m) ⁻¹vs log₁₀ (concentration). Borer, P. N., Dengler, B., Tinoco, I., Jr.,and Uhlenbeck, O. C., J. Mol. Biol., 1974, 86, 843-853.

EXAMPLE 7 Gel Shift Assay

The structured ras target transcript, a 47-nucleotide hairpin containingthe mutated codon 12, was prepared and mapped as described in Lima etal., Biochemistry 1992, 31, 12055-12061. Hybridization reactions wereprepared in 20 μl containing 100 mM sodium, 10 mM phosphate, 0.1 mMEDTA, 100 CPM of T7-generated RNA (approximately 10 pM), and antisenseoligonucleotide ranging in concentration from 1 pM to 10 μM. Reactionswere incubated 24 hours at 37° C. Following hybridization, loadingbuffer was added to the reactions and reaction products were resolved on20% native polyacrylamide gels, prepared using 45 mM tris-borate and 1mM MgCl₂ (TBM). Electrophoresis was carried out at 10° C. and gels werequantitated using a Molecular Dynamics Phosphorimager.

EXAMPLE 8 RNase H Analysis

RNase H assays were performed using a chemically synthesized 25-baseoligoribonucleotide corresponding to bases +23 to +47 of activated(codon 12, G→U) H-ras mRNA. The 5' end-labeled RNA was used at aconcentration of 20 nM and incubated with a 10-fold molar excess ofantisense oligonucleotide in a reaction containing 20 mM Tris-Cl, pH7.5, 100 mM KCl, 10 mM MgCl₂, 1 mM dithiothreitol, 10 μg tRNA and 4 URNasin in a final volume of 10 μl. The reaction components werepreannealed at 37° C. for 15 minutes then allowed to cool slowly to roomtemperature. HeLa cell nuclear extracts were used as a source ofmammalian RNase H. Reactions were initiated by addition of 2 μg ofnuclear extract (5 μl) and reactions were allowed to proceed for 10minutes at 37° C. Reactions were stopped by phenol/chloroform extractionand RNA components were precipitated with ethanol. Equal CPMs wereloaded on a 20% polyacrylamide gel containing 7M urea and RNA cleavageproducts were resolved and visualized by electrophoresis followed byautoradiography. Quantitation of cleavage products was performed using aMolecular Dynamics Densitometer.

EXAMPLE 9 ras Transactivation Reporter Gene System

The expression plasmid pSV2-oli, containing an activated (codon 12,GGC→GTC) H-ras cDNA insert under control of the constitutive SV40promoter, was a gift from Dr. Bruno Tocque (Rhone-Poulenc Sante, Vitry,France) . This plasmid was used as a template to construct, by PCR, aH-ras expression plasmid under regulation of the steroid-inducible mousemammary tumor virus (MMTV) promoter. To obtain H-ras coding sequences,the 570 bp coding region of the H-ras gene was amplified by PCR. The PCRprimers were designed with unique restriction endonuclease sites intheir 5'-regions to facilitate cloning. The PCR product containing thecoding region of the H-ras codon 12 mutant oncogene was gel purified,digested, and gel purified once again prior to cloning. Thisconstruction was completed by cloning the insert into the expressionplasmid pMAMneo (Clontech Laboratories, Calif.).

The ras-responsive reporter gene pRDO53 was used to detect rasexpression. Owen et al., Proc. Natl. Acad. Sci. U.S.A. 1990, 87,3866-3870.

EXAMPLE 10 Northern Blot Analysis of ras Expression in vivo

The human urinary bladder cancer cell line T24 was obtained from theAmerican Type Culture Collection (Rockville Md.). Cells were grown inMcCoy's 5A medium with L-glutamine (Gibco BRL, Gaithersburg Md.),supplemented with 10% heat-inactivated fetal calf serum and 50 U/ml eachof penicillin and streptomycin. Cells were seeded on 100 mm plates. Whenthey reached 70% confluency, they were treated with oligonucleotide.Plates were washed with 10 ml prewarmed PBS and 5 ml of Opti-MEMreduced-serum medium containing 2.5 μl DOTMA was added. Oligonucleotidewas then added to the desired concentration. After 4 hours of treatment,the medium was replaced with McCoy's medium. Cells were harvested 48hours after oligonucleotide treatment and RNA was isolated using astandard CsCl purification method. Kingston, R. E., in Current Protocolsin Molecular Biology, (F. M. Ausubel, R. Brent, R. E. Kingston, D. D.Moore, J. A. Smith, J. G. Seidman and K. Strahl, eds.), John Wiley andSons, NY.

The human epithelioid carcinoma cell line HeLa 229 was obtained from theAmerican Type Culture Collection (Bethesda, Md.). HeLa cells weremaintained as monolayers on 6-well plates in Dulbecco's Modified Eagle'smedium (DMEM) supplemented with 10% fetal bovine serum and 100 U/mlpenicillin. Treatment with oligonucleotide and isolation of RNA wereessentially as described above for T24 cells.

Northern hybridization: 10 μg of each RNA was electrophoresed on a 1.2%agarose/formaldehyde gel and transferred overnight to GeneBind 45 nylonmembrane (Pharmacia LKB, Piscataway, N.J.) using standard methods.Kingston, R. E., in Current Protocols in Molecular Biology, (F. M.Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G.Seidman and K. Strahl, eds.), John Wiley and Sons, NY. RNA wasUV-crosslinked to the membrane. Double-stranded ³² P-labeled probes weresynthesized using the Prime a Gene labeling kit (Promega, Madison Wis.).The ras probe was a SalI-NheI fragment of a CDNA clone of the activated(mutant) H-ras mRNA having a GGC-to-GTC mutation at codon-12. Thecontrol probe was G3PDH. Blots were prehybridized for 15 minutes at 68°C. with the QuickHyb hybridization solution (Stratagene, La Jolla,Calif.). The heat-denatured radioactive probe (2.5×10⁶ counts/2 mlhybridization solution) mixed with 100 μl of 10 mg/ml salmon sperm DNAwas added and the membrane was hybridized for 1 hour at 68° C. The blotswere washed twice for 15 minutes at room temperature in 2x SSC/0.1% SDSand once for 30 minutes at 60° C. with 0.1XSSC/0.1% SDS. Blots wereautoradiographed and the intensity of signal was quantitated using anImageQuant PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).Northern blots were first hybridized with the ras probe, then strippedby boiling for 15 minutes in 0.1x SSC/0.1%SDS and rehybridized with thecontrol G3PDH probe to check for correct sample loading.

EXAMPLE 11 Antisense Oligonucleotide Inhibition of Proliferation ofCancer Cells

Cells were cultured and treated with oligonucleotide essentially asdescribed in Example 10. Cells were seeded on 60 mm plates and weretreated with oligonucleotide in the presence of DOTMA when they reached70% confluency.

Time course experiment: On day 1, cells were treated with a single doseof oligonucleotide at a final concentration of 100 nM. The growth mediumwas changed once on day 3 and cells were counted every day for 5 days,using a counting chamber.

Dose-response experiment: Various concentrations of oligonucleotide (10,25, 50, 100 or 250 nM) were added to the cells and cells were harvestedand counted 3 days later. Oligonucleotides 2570, 3985 and 4690 weretested for effects on T24 cancer cell proliferation.

EXAMPLE 12 Synthesis of 2-(amino)adenine-substituted Oligonucleotides

Oligonucleotides are synthesized as in Example 1, with the followingexception: at positions at which a 2(amino)adenine is desired, thestandard phosphoramidite is replaced with a commercially available2-aminodeoxyadenosine phosphoramidite (Chemgenes).

EXAMPLE 13 Culture of A549 cells

A549 cells (obtained from the American Type Culture Collection, BethesdaMd.) were grown to confluence in 6-well plates (Falcon Labware, LincolnPark, N.J.) in Dulbecco's modified Eagle's medium (DME) containing 1 gglucose/liter and 10% fetal calf serum (FCS, Irvine Scientific, SantaAna, Calif.).

EXAMPLE 14 Oligonucleotide Treatment of Human Tumor Cells in Nude MiceIntraperitoneal Injection

Human lung carcinoma A549 cells were harvested and 5×10⁶ cells (200 μl)were injected subcutaneously into the inner thigh of nude mice. Palpabletumors develop in approximately one month. Phosphorothioateoligonucleotides ISIS 2503 and 1082 (unrelated control) wereadministered to mice intraperitoneally at a dosage of 20 mg/kg bodyweight, every other day for approximately ten weeks. Mice were monitoredfor tumor growth during this time.

EXAMPLE 15 Oligonucleotide Treatment of Human Tumor Cells in Nude MiceSubcutaneous Injection with Cationic Lipid

Human lung carcinoma A549 cells were harvested and 5×10⁶ cells (200 μl)were injected subcutaneously into the inner thigh of nude mice. Palpabletumors develop in approximately one month. Phosphorothioateoligonucleotides ISIS 2503 and the unrelated control oligonucleotide1082 (dosage 5 mg/kg), prepared in a cationic lipid formulation(DMRIE/DOPE, 60 mg/kg) were administered to mice subcutaneously at thetumor site. Drug treatment began one week following tumor cellinoculation and was given twice a week for only four weeks. Mice weremonitored for tumor growth for a total of nine weeks.

EXAMPLE 16 Stability of 2' Modified Oligonucleotides in T24 Cells

T24 bladder cancer cells were grown as described in Example 10. Cellswere treated with a single dose (1 μM) of oligonucleotide and assayedfor H-ras mRNA expression by Northern blot analysis 24 hours later.Oligonucleotides tested were analogs of ISIS 2570 (SEQ ID NO: 3), a 17mer targeted to H-ras codon 12.

EXAMPLE 17 Activity of Ki-ras Oligonucleotides Against Three ColonCarcinoma Cell Lines

Human colon carcinoma cell lines Calu 1, SW480 and SW620 were obtainedfrom the American Type Culture Collection (ATCC) and cultured andmaintained as described for HeLa cells in Example 10. Cells were treatedwith a single dose of oligonucleotide (200 mM) and Ki-ras mRNAexpression was measured by Northern blot analysis 24 hours later. Forproliferation studies, cells were treated with a single dose ofoligonucleotide (200 nM) at day zero and cell number was monitored overa five-day period.

EXAMPLE 18 Oligonucleotide Inhibition of Mutant vs. Wild-type Ki-ras

SW480 cells were cultured as in the previous example. HeLa cells werecultured as in Example 10. Cells were treated with a single dose (100nM) of oligonucleotide and mRNA levels were determined by Northern blotanalysis 24 hours later.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 55    (2) INFORMATION FOR SEQ ID NO: 1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    CTTATATTCCGTCATCGCTC20    (2) INFORMATION FOR SEQ ID NO: 2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    TCCGTCATCGCTCCTCAGGG20    (2) INFORMATION FOR SEQ ID NO: 3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:    CCACACCGACGGCGCCC17    (2) INFORMATION FOR SEQ ID NO: 4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:    CCCACACCGACGGCGCCCA19    (2) INFORMATION FOR SEQ ID NO: 5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:    GCCCACACCGACGGCGCCCAC21    (2) INFORMATION FOR SEQ ID NO: 6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:    TGCCCACACCGACGGCGCCCACC23    (2) INFORMATION FOR SEQ ID NO: 7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:    TATTCCGTCATCGCTCCTCA20    (2) INFORMATION FOR SEQ ID NO: 8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 5    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:    CGACG5    (2) INFORMATION FOR SEQ ID NO: 9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:    CCGACGG7    (2) INFORMATION FOR SEQ ID NO: 10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:    ACCGACGGC9    (2) INFORMATION FOR SEQ ID NO: 11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:    CACCGACGGCG11    (2) INFORMATION FOR SEQ ID NO: 12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:    ACACCGACGGCGC13    (2) INFORMATION FOR SEQ ID NO: 13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:    CACACCGACGGCGCC15    (2) INFORMATION FOR SEQ ID NO: 14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:    CCACACCGACGGCGCC16    (2) INFORMATION FOR SEQ ID NO: 15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:    CACACCGACGGCGCCC16    (2) INFORMATION FOR SEQ ID NO: 16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:    CCCACACCGACGGCGCCC18    (2) INFORMATION FOR SEQ ID NO: 17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:    CCACACCGACGGCGCCCA18    (2) INFORMATION FOR SEQ ID NO: 18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:    TTGCCCACACCGACGGCGCCCACCA25    (2) INFORMATION FOR SEQ ID NO: 19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:    CCACACCGCCGGCGCCC17    (2) INFORMATION FOR SEQ ID NO: 20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:    CTGCCTCCGCCGCCGCGGCC20    (2) INFORMATION FOR SEQ ID NO: 21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:    CAGTGCCTGCGCCGCGCTCG20    (2) INFORMATION FOR SEQ ID NO: 22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:    AGGCCTCTCTCCCGCACCTG20    (2) INFORMATION FOR SEQ ID NO: 23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:    TTCAGTCATTTTCAGCAGGC20    (2) INFORMATION FOR SEQ ID NO: 24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:    TTATATTCAGTCATTTTCAG20    (2) INFORMATION FOR SEQ ID NO: 25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:    CAAGTTTATATTCAGTCATT20    (2) INFORMATION FOR SEQ ID NO: 26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:    GCCTACGCCACCAGCTCCAAC21    (2) INFORMATION FOR SEQ ID NO: 27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:    CTACGCCACCAGCTCCA17    (2) INFORMATION FOR SEQ ID NO: 28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:    GTACTCCTCTTGACCTGCTGT21    (2) INFORMATION FOR SEQ ID NO: 29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:    CCTGTAGGAATCCTCTATTGT21    (2) INFORMATION FOR SEQ ID NO: 30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:    GGTAATGCTAAAACAAATGC20    (2) INFORMATION FOR SEQ ID NO: 31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:    GGAATACTGGCACTTCGAGG20    (2) INFORMATION FOR SEQ ID NO: 32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:    TACGCCAACAGCTCC15    (2) INFORMATION FOR SEQ ID NO: 33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:    TTTTCAGCAGGCCTCTCTCC20    (2) INFORMATION FOR SEQ ID NO: 34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:    TCAGTAATAGCCCCACATGG20    (2) INFORMATION FOR SEQ ID NO: 35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:    CCGGGTCCTAGAAGCTGCAG20    (2) INFORMATION FOR SEQ ID NO: 36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:    TAAATCAGTAAAAGAAACCG20    (2) INFORMATION FOR SEQ ID NO: 37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:    GGACACAGTAACCAGGCGGC20    (2) INFORMATION FOR SEQ ID NO: 38:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:    AACAGAAGCTACACCAAGGG20    (2) INFORMATION FOR SEQ ID NO: 39:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:    CAGACCCATCCATTCCCGTG20    (2) INFORMATION FOR SEQ ID NO: 40:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:    GCCAAGAAATCAGACCCATC20    (2) INFORMATION FOR SEQ ID NO: 41:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:    AGGGGGAAGATAAAACCGCC20    (2) INFORMATION FOR SEQ ID NO: 42:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:    CGCTTCCATTCTTTCGCCAT20    (2) INFORMATION FOR SEQ ID NO: 43:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:    CCGCACCCAGACCCGCCCCT20    (2) INFORMATION FOR SEQ ID NO: 44:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:    CAGCCCCCACCAAGGAGCGG20    (2) INFORMATION FOR SEQ ID NO: 45:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:    GTCATTTCACACCAGCAAGA20    (2) INFORMATION FOR SEQ ID NO: 46:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:    CAGTCATTTCACACCAGCAA20    (2) INFORMATION FOR SEQ ID NO: 47:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:    CTCAGTCATTTCACACCAGC20    (2) INFORMATION FOR SEQ ID NO: 48:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:    CGTGGGCTTGTTTTGTATCA20    (2) INFORMATION FOR SEQ ID NO: 49:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:    CCATACAACCCTGAGTCCCA20    (2) INFORMATION FOR SEQ ID NO: 50:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:    CAGACAGCCAAGTGAGGAGG20    (2) INFORMATION FOR SEQ ID NO: 51:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:    CCAGGGCAGAAAAATAACAG20    (2) INFORMATION FOR SEQ ID NO: 52:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:    TTTGTGCTGTGGAAGAACCC20    (2) INFORMATION FOR SEQ ID NO: 53:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:    GCTATTAAATAACAATGCAC20    (2) INFORMATION FOR SEQ ID NO: 54:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:    ACTGATCACAGCTATTAAAT20    (2) INFORMATION FOR SEQ ID NO: 55:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21    (B) TYPE: Nucleic Acid    (C) STRANDEDNESS: Single    (D) TOPOLOGY: Linear    (iv) ANTI-SENSE: Yes    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:    GCCGAGGTCCATGTCGTACGC21    __________________________________________________________________________

What is claimed is:
 1. An oligonucleotide 8 to 30 nucleotides in lengthwhich is targeted to a nucleic acid encoding human N-ras and which iscapable of inhibiting ras expression, wherein said oligonucleotidecomprises SEQ ID NO: 44, 45, 46, 47, 49 or
 52. 2. The oligonucleotide ofclaim 1 which comprises at least one backbone modification.
 3. Theoligonucleotide of claim 1 wherein at least one of the nucleotide unitsof the oligonucleotide is modified at the 2' position of the sugar. 4.The oligonucleotide of claim 1 which is a chimeric oligonucleotide. 5.The oligonucleotide of claim 1 in a pharmaceutically acceptable carrier.6. A method of modulating the expression of human ras comprisingcontacting tissues or cells containing a human ras gene with aneffective amount of an oligonucleotide of claim 1, whereby expression ofras is modulated.
 7. A method of inhibiting-the proliferation of cancercells comprising contacting cancer cells with an effective amount of anoligonucleotide of claim 1, whereby proliferation of the cancer cells isinhibited.
 8. A method of preventing or treating a condition arisingfrom the activation of a ras oncogene comprising contacting an animalsuspected of having a condition arising from the activation of a rasoncogene with an effective amount of an oligonucleotide of claim 1,whereby said condition is prevented or treated.